WO2018160540A1 - Therapeutic rna - Google Patents

Therapeutic rna Download PDF

Info

Publication number: WO2018160540A1
Authority: WO; WIPO (PCT)
Prior art keywords: rna; seq; tumor; protein; identity
Prior art date: 2017-02-28

Application number

PCT/US2018/019878

Other languages

English (en)

French (fr)

Inventor

Friederike GIESEKE

Ugur Sahin

Dmitri G. Wiederschain

Timothy R. WAGENAAR

Original Assignee

Sanofi

Biontech Rna Pharmaceuticals Gmbh

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2017-02-28

Filing date

2018-02-27

Publication date

2018-09-07

2018-02-27 Priority to KR1020197028213A priority Critical patent/KR102700956B1/ko

2018-02-27 Priority to EA201992022A priority patent/EA201992022A1/ru

2018-02-27 Priority to PE2019001764A priority patent/PE20200735A1/es

2018-02-27 Priority to AU2018229278A priority patent/AU2018229278A1/en

2018-02-27 Priority to JP2019546924A priority patent/JP6949131B2/ja

2018-02-27 Priority to MX2019010269A priority patent/MX2019010269A/es

2018-02-27 Priority to SG11201907846VA priority patent/SG11201907846VA/en

2018-02-27 Priority to EP18709243.2A priority patent/EP3589308A1/en

2018-02-27 Priority to CA3054733A priority patent/CA3054733A1/en

2018-02-27 Priority to CN201880013857.3A priority patent/CN110337305A/zh

2018-02-27 Priority to BR112019017743A priority patent/BR112019017743A2/pt

2018-02-27 Priority to CR20190444A priority patent/CR20190444A/es

2018-02-27 Application filed by Sanofi, Biontech Rna Pharmaceuticals Gmbh filed Critical Sanofi

2018-02-27 Priority to KR1020247028726A priority patent/KR20240144261A/ko

2018-09-07 Publication of WO2018160540A1 publication Critical patent/WO2018160540A1/en

2019-08-25 Priority to IL26889419A priority patent/IL268894A/he

2019-08-27 Priority to US16/552,248 priority patent/US20200147176A1/en

2019-08-27 Priority to PH12019501959A priority patent/PH12019501959A1/en

2019-09-25 Priority to CONC2019/0010355A priority patent/CO2019010355A2/es

2021-04-30 Priority to US17/245,605 priority patent/US11865159B2/en

2023-11-21 Priority to US18/516,006 priority patent/US20240173382A1/en

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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/212—IFN-alpha
- A—HUMAN NECESSITIES
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5434—IL-12
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- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5443—IL-15
- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/102—Mutagenizing nucleic acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/33—Chemical structure of the base
- C12N2310/335—Modified T or U

Definitions

Solid tumors include benign and malignant
cancer cancer is often divided into two main types: solid tumor cancer and hematological (blood) cancers. It is estimated that more than 1.5 million cases of cancer are diagnosed in the United States each year, and more than 500,000 people in the United States will die each year from cancer.
Solid tumor cancers are particularly difficult to treat.
Current treatments include surgery, radiotherapy, immunotherapy and chemotherapy.
Surgery alone may be an appropriate treatment for small localized tumors, but large invasive tumors and most metastatic malignancies are usually unresectable by surgery.
Other common treatments such as radiotherapy and chemotherapy are associated with undesirable side effects and damage to healthy cells.
compositions, uses, and methods that can overcome present shortcomings in treatment of solid tumors.
Administration of therapeutic RNAs disclosed herein can reduce tumor size, extend survival time, and/or protect against metastasis and/or recurrence of the tumor.
Embodiment 1 A composition comprising RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14 and RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27.
Embodiment 2 The composition of embodiment 1, further comprising RNA encoding an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24.
Embodiment 3 The composition of embodiment 1, further comprising RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9.
Embodiment 4 The composition of embodiment 1, further comprising RNA encoding an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19.
Embodiment 5 A composition comprising:
RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14;
RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27; and c. RNA encoding an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19.
Embodiment 6 The composition of embodiment 5, further comprising RNA encoding an IL-15sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24.
Embodiment 7 The composition of embodiment 5, further comprising RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9.
Embodiment 8 The composition of any one of embodiments 1-7, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment 9 The composition of any one of embodiments 1-7, wherein each RNA comprises a modified nucleobase in place of each uridine.
Embodiment 10 The composition of any one of embodiments 8-9, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl- uridine (m 5 U).
the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl- uridine (m 5 U).
Embodiment 11 The composition of embodiment 10, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
Embodiment 12 The composition of any one of embodiments 1-11, wherein at least one RNA further comprises a 5’ cap.
Embodiment 13 The composition of any one of embodiments 1-11, wherein each RNA further comprises a 5’ cap.
Embodiment 14 The composition of any one of embodiments 12-13, wherein the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 15 The composition of any one of embodiments 1-14, wherein at least one RNA further comprises a 5’ UTR.
Embodiment 16 The composition of any one of embodiments 1-14, wherein each RNA further comprises a 5’ UTR.
Embodiment 17 The composition of any one of embodiments 15-16, wherein the 5’ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.
Embodiment 18 The composition of any one of embodiments 1-17, wherein at least one RNA further comprises a 3’ UTR.
Embodiment 19 The composition of any one of embodiments 1-17, wherein each RNA further comprises a 3’ UTR.
Embodiment 20 The composition of any one of embodiments 18-19, wherein the 3’ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
Embodiment 21 The composition of any one of embodiments 1-20, wherein at least one RNA further comprises a poly-A tail.
Embodiment 22 The composition of any one of embodiments 1-20, wherein each RNA further comprises a poly-A tail.
Embodiment 23 The composition of any one of embodiments 21-22, wherein the poly- A tail comprises at least 100 nucleotides.
Embodiment 24 The composition of any one of embodiments 1-23, wherein at least one RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
Embodiment 25 The composition of any one of embodiments 1-23, wherein each RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
Embodiment 26 The composition of any one of embodiments 24-25, wherein
the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G;
the 5’ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6;
the 3’ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8;
the poly-A tail comprises at least 100 nucleotides.
Embodiment 27 A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering the composition of any one of embodiments 1-26 to the subject.
Embodiment 28 A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering to the subject:
RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 17 or 18; and b.
RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 29, thereby treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in the subject.
Embodiment 29 The method of embodiment 28, further comprising administering RNA encoding an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 30 The method of embodiment 28, further comprising administering RNA encoding an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 26.
Embodiment 31 Embodiment 31.
RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 32 The method of embodiment 28, further comprising administering RNA encoding
an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 26; and
an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 33 The method of embodiment 28, further comprising administering RNA encoding
an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 12 or 13; and
an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 34 The method of any one of embodiments 27-33, wherein the cancer is a sarcoma, carcinoma, or lymphoma.
Embodiment 35 The method of any one of embodiments 27-33, wherein the cancer is a solid tumor.
Embodiment 36 The method of embodiment 35, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
Embodiment 37 The method of embodiment 35, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma, testicular tumor, carcinoma, adenocarcinoma, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
HL Hodgkin lymphoma
non-Hodgkin lymphoma prostate tumor
ovarian tumor renal cell tumor
renal cell tumor gastrointestinal tract tumor
hepatic tumor colorectal tumor
tumor with vasculature mesothelioma tumor
pancreatic tumor breast tumor
Embodiment 38 The method of any one of embodiments 27-37, wherein the
composition is administered intra-tumorally or peri-tumorally.
Embodiment 39 The method of embodiment 38, wherein the injected tumor and a non- injected tumor are both reduced in size after intra- or peri- tumoral injection into or near the first tumor.
Embodiment 40 The method of any one of embodiments 27-39, wherein the subject is human.
Embodiment 41 The method of any one of embodiments 27-40, wherein another
Embodiment 42 The method of embodiment 41, wherein the other therapy is surgery to excise, resect, or debulk the tumor.
Embodiment 43 The method of embodiment 41, wherein the other therapy is
Embodiment 44 The method of any one of embodiments 27-43, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment 45 The method of any one of embodiments 27-43, wherein each RNA comprises a modified nucleobase in place of each uridine.
Embodiment 46 The method of any one of embodiments 44-45, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl- uridine (m 5 U).
Embodiment 47 The method of embodiment 46, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
Embodiment 48 The method of any one of embodiments 27-47, wherein at least one RNA further comprises a 5’ cap.
Embodiment 49 The method of any one of embodiments 27-47, wherein each RNA further comprises a 5’ cap.
Embodiment 50 The method of any one of embodiments 48-49, wherein the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 51 The method of any one of embodiments 27-50, wherein at least one RNA further comprises a 5’ UTR.
Embodiment 52 The method of any one of embodiments 27-50, wherein each RNA further comprises a 5’ UTR.
Embodiment 53 The method of any one of embodiments 51-52, wherein the 5’ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.
Embodiment 54 The method of any one of embodiments 27-54, wherein at least one RNA further comprises a 3’ UTR.
Embodiment 55 The method of any one of embodiments 27-54, wherein each RNA further comprises a 3’ UTR.
Embodiment 56 The method of any one of embodiments 54-55, wherein the 3’ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
Embodiment 57 The method of any one of embodiments 27-56, wherein at least one RNA further comprises a poly-A tail.
Embodiment 58 The method of any one of embodiments 27-56, wherein each RNA further comprises a poly-A tail.
Embodiment 59 The method of any one of embodiments 57-58, wherein the poly-A tail comprises at least 100 nucleotides.
Embodiment 60 The method of any one of embodiments 27-59, wherein at least one RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
Embodiment 61 The method of any one of embodiments 27-59, wherein each RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
Embodiment 62 The method of any one of embodiments 60-61, wherein
the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G;
the 5’ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6;
the 3’ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8;
the poly-A tail comprises at least 100 nucleotides.
Embodiment 63 A codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 11.
Embodiment 64 The DNA of embodiment 63, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 11.
Embodiment 65 A codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 13.
Embodiment 66 The RNA of embodiment 65, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 13.
Embodiment 67 An RNA produced from the DNA of any one of embodiments 63 or 64.
Embodiment 68 A codon-optimized DNA comprising or consisting of:
nucleotides encoding a linker between the nucleotides of a) and b).
Embodiment 69 The DNA of embodiment 68, wherein the linker comprises nucleotides 985-1026 of SEQ ID NO: 16.
Embodiment 70 The DNA of any one of embodiments 68 and 69, wherein part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 16; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 16.
a codon-optimized RNA comprising or consisting of:
nucleotides having at least 81% identity to nucleotides 1027-1623 of SEQ ID NO: 18; and c. nucleotides encoding a linker between the nucleotides of a) and b).
Embodiment 72 The RNA of embodiment 71, wherein the linker comprises nucleotides 985-1026 of SEQ ID NO: 18.
Embodiment 73 The RNA of any one of embodiments 71 and 72, wherein part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 18; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 18.
Embodiment 74 An RNA produced from the DNA of any one of embodiments 68-70.
Embodiment 75 A codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 21.
Embodiment 76 The DNA of embodiment 75, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 21.
Embodiment 77 A codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 23.
Embodiment 78 The RNA of embodiment 77, comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 23.
Embodiment 79 An RNA produced from the DNA of any one of embodiments 75-76.
Embodiment 80. A DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1-321 of SEQ ID NO: 25;
contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 382-729 of SEQ ID NO: 25;
nucleotides encoding a linker between the nucleotides of a) and b).
Embodiment 81 An RNA produced from the DNA of embodiment 80.
Embodiment 82 A RNA comprising or consisting of:
contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1-321 of SEQ ID NO: 26; b. contiguous nucleotides comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 382-729 of SEQ ID NO: 26; and
nucleotides encoding a linker between the nucleotides of a) and b).
Embodiment 83 A DNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 28.
Embodiment 84 An RNA produced from the DNA of embodiment 83.
Embodiment 85 An RNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 29.
Embodiment 86 The RNA of any one of embodiments 67, 74, 79, 81, and 84 wherein the RNA is transcribed from the DNA in vitro.
Embodiment 87 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-85, wherein at least one uridine is replaced with a modified nucleobase.
Embodiment 88 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-85, wherein each uridine is replaced with a modified nucleobase.
Embodiment 89 The RNA of embodiment 87 or 88, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
Embodiment 90 The RNA of embodiment 89, wherein the modified nucleobase is N1- methyl-pseudouridine (m 1 ⁇ ).
Embodiment 91 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-90, further comprising a 5’ cap.
Embodiment 92 The RNA of embodiment 91, wherein the 5’ cap is m2 7,3’-O Gppp(m1 2’- O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 93 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-92, further comprising a 5’ UTR.
Embodiment 94 The RNA of embodiment 93, wherein the 5’ UTR comprises or
Embodiment 95 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-94, further comprising a 3’ UTR.
Embodiment 96 The RNA of embodiment 95, wherein the 3’ UTR comprises or
Embodiment 97 consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
Embodiment 97 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-96, further comprising a poly-A tail.
Embodiment 98 The RNA of embodiment 97, wherein the poly-A tail comprises at least 100 nucleotides.
Embodiment 99 The RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-97, further comprising a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
Embodiment 100 The RNA of embodiment 99, wherein
the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G;
the 5’ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6;
the 3’ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8 and;
the poly-A tail comprises at least 100 nucleotides.
a DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11;
nucleotides of parts a) and b) form a single transcript.
a DNA comprising or consisting of:
nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1- 984 of SEQ ID NO: 16;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21;
nucleotides of part a) and b) form a single transcript.
Embodiment 104 A DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28;
nucleotides of part a) and b) form a single transcript.
Embodiment 105 A DNA comprising or consisting of:
nucleotides 1-321 of SEQ ID NO: 25 a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382- 729 of SEQ ID NO: 25;
nucleotides of part a), b), and c) form a single transcript.
a DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11;
a DNA comprising or consisting of:
nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1- 984 of SEQ ID NO: 16;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16;
nucleotides of part a), b) and c) form a single transcript.
a DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21;
nucleotides of part a) and b) form a single transcript.
a DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28;
nucleotides of part a) and b) form a single transcript.
Embodiment 110 A DNA comprising or consisting of:
nucleotides 1-321 of SEQ ID NO: 25 a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25;
nucleotides 382- 729 of SEQ ID NO: 25 b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382- 729 of SEQ ID NO: 25; and c. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), and c) form a single transcript.
Embodiment 111 A DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 83% identity to SEQ ID NO: 11; b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and
nucleotides of part a), b), and c) form a single transcript.
Embodiment 112. A DNA comprising or consisting of:
nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 78% identity to nucleotides 1- 984 of SEQ ID NO: 16;
nucleotides 1027-1623 of SEQ ID NO: 16 b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or 81% identity to nucleotides 1027-1623 of SEQ ID NO: 16;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), c), and d) form a single transcript.
a DNA comprising or consisting of:
nucleotides of part b a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 21; b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5, wherein the nucleotides of part b) regulate the expression of the nucleotides of part a); and
nucleotides of part a), b) and c) form a single transcript.
Embodiment 114 A DNA comprising or consisting of:
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 28; b. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5; and
nucleotides of part a), b), and c) form a single transcript.
Embodiment 115 A DNA comprising or consisting of:
nucleotides 1-321 of SEQ ID NO: 25 a. contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 1-321 of SEQ ID NO: 25;
nucleotides 382- 729 of SEQ ID NO: 25 comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotides 382- 729 of SEQ ID NO: 25;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NOs: 1, 3, or 5;
contiguous nucleotides comprising or consisting of nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 7, wherein when transcribed, the nucleotides of part a), b), c), and d) form a single transcript.
Embodiment 116 An RNA produced from any one of the DNAs of embodiments 101-115.
Embodiment 117 The RNA of embodiment 116, wherein at least one uridine is replaced with a modified nucleobase.
Embodiment 118 The RNA of embodiment 116, wherein each uridine is replaced with a modified nucleobase.
Embodiment 119 The RNA of any one of embodiments 117-118, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5- methyl-uridine (m 5 U).
Embodiment 120 The RNA of embodiment 119, wherein the modified
nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
Embodiment 121 The RNA of any one of embodiments 116-120, wherein the RNA further comprises a 5’ cap.
Embodiment 122 The RNA of embodiment 121, wherein the 5’ cap is m 2 7,3’- O Gppp(m1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 123 The RNA of any of embodiments 116-122, wherein the RNA is substantially free of double-stranded RNA.
Embodiment 124 The RNA of any of embodiments 116-122, wherein double stranded RNA has been removed from the RNA.
Embodiment 125 The RNA of any one of embodiments 123-124, wherein the RNA has been purified via HPLC or cellulose-based chromatography.
Embodiment 126 A pharmaceutical formulation comprising any one of the DNA or RNAs of embodiments 63-125 and a pharmaceutically acceptable excipient.
Embodiment 127 A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one or more of the RNAs or DNAs of embodiments 63-125, or the pharmaceutical formulation of embodiment 126.
Embodiment 128 A method of producing a polypeptide encoding IL-2, IL-12sc, IL-15 sushi, GM-CSF and IFN ⁇ 2b in vivo comprising administering to a subject one or more of the DNAs or RNAs of any one of embodiment 63-125, the composition of any one of embodiments 1-26, or the pharmaceutical formulation of embodiment 126.
Embodiment 129 A composition comprising at least two RNAs, wherein the RNAs encode different proteins, and wherein the RNAs are selected from the RNAs of any one of embodiments 65-67, 71-74, 78-79, 81-82, 84-100, and 116-125.
Embodiment 130 The composition of embodiment 129, wherein the composition comprises two RNAs encoding GM-CSF and IL-12sc.
Embodiment 131 The composition of embodiment 129, wherein the composition comprises three RNAs encoding GM-CSF, IL-12sc, and IFN ⁇ 2b.
Embodiment 132 The composition of embodiment 129, wherein the composition comprises three RNAs encoding GM-CSF, IL-2, and IFN ⁇ 2b.
Embodiment 133 The composition of embodiment 129, wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-2 and IFN ⁇ 2b.
Embodiment 134 The composition of embodiment 129, wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-15 sushi and IFN ⁇ 2b.
Embodiment 135. The composition of any one of embodiments 130-134, wherein the RNA encoding GM-CSF is selected from the RNA of any one of embodiments 84-85.
Embodiment 136 The composition of any one of embodiments 130-134, wherein the RNA encoding IL-12sc is selected from the RNA of any one of embodiments 71- 74.
Embodiment 137 The composition of any one of embodiments 131-134, wherein the RNA encoding IFN ⁇ 2b is selected from the RNA of any one of embodiments 77- 79.
Embodiment 138 The composition of embodiment 134, wherein the RNA
Embodiment 140 A method of treating or preventing solid tumor cancer
RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); and c. an RNA comprising an RNA encoding IFN ⁇ 2b (SEQ ID NOs: 22 or 23), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 141 A method of treating or preventing solid tumor cancer
RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); c. an RNA comprising an RNA encoding IFN ⁇ 2b (SEQ ID NOs: 22 or 23); and d. an RNA comprising an RNA encoding IL-2 (SEQ ID NOs: 12 or 13), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 142 A method of treating or preventing solid tumor cancer
RNA comprising an RNA encoding IL-12sc (SEQ ID NOs: 17 or 18); b. an RNA comprising an RNA encoding GM-CSF (SEQ ID NO: 29); c. an RNA comprising an RNA encoding IFN ⁇ 2b (SEQ ID NOs: 22 or 23); and d. an RNA comprising an RNA encoding IL-15 sushi (SEQ ID NO: 26), wherein each RNA comprises a modified nucleobase in place of each uridine, and wherein each RNA comprises a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 143 A composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acids of SEQ ID NO: 9, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 144 A composition comprising an RNA encoding an IL-12sc
each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 145 A composition comprising an RNA encoding a GM-CSF protein having at least 95% identity to the amino acids of SEQ ID NO: 27, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 146 A composition comprising an RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acids of SEQ ID NO: 19, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 147 A composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acids of SEQ ID NO: 24, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 148 An IL-12sc RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 149 Embodiment 149.
a GM-CSF RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 150 An IFN ⁇ 2b RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a uridine analog, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 151 An IL-15 sushi RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 152 An IL-2 RNA composition comprising or consisting of
Embodiment 153 Embodiment 153.
An IL-12sc RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 154 A GM-CSF RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 155 An IFN ⁇ 2b RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 156 An IL-15 sushi RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 157 An IL-2 RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 12 or 13, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5’ UTR (SEQ ID NOs: 2, 4, or 6), a 3’ UTR (SEQ ID NO: 8), a 5’ cap, and a poly-A tail.
Embodiment 158 The composition of any of embodiments 143-157, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5- methyl-uridine (m 5 U).
Embodiment 159 A composition comprising an RNA encoding IFN ⁇ 2b, wherein the RNA is altered to have reduced immunogenicity as compared to un-altered RNA.
Embodiment 160 The composition of embodiment 159, wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.
Embodiment 161 The composition of embodiment 159, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl- uridine (m 5 U).
Embodiment 162 The composition of embodiment 161, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
Embodiment 163 The composition of any one of embodiments 159-162, wherein the alteration comprises a reduction in the amount of double-stranded RNA.
Embodiment 164 The composition of embodiment 163, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.
Embodiment 165 The composition of any one of embodiments 159-164, wherein the alteration reduces RNA recognition by an innate immune system as compared to un-altered RNA.
Embodiment 166 The composition of any one of embodiments 159-165, wherein the alteration comprises addition of a 5’ cap to the RNA.
Embodiment 167 The composition of embodiment 166, wherein the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 168 The composition of any one of embodiments 159-167, further comprising a second RNA encoding a peptide or protein of interest.
Embodiment 169 The composition of embodiment 168, wherein the peptide or protein of interest is a peptide or protein selected or derived from cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, ⁇ -catenin inhibitors, activators of the STING pathway, activators of the retinoic inducible gene (RIG)-I pathway, agonists of toll-like receptor (TLR) pathways, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis.
Embodiment 170 The composition of embodiment 168 or 169, wherein the second RNA is altered to have reduced immunogenicity as compared to un-altered RNA.
Embodiment 171 The composition of embodiment 170, wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.
Embodiment 172 The composition of embodiment 171, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl- uridine (m 5 U).
Embodiment 173 The composition of embodiment 172, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
Embodiment 174 The composition of any one of embodiments 168-173, wherein the alteration comprises a reduction in the amount of double-stranded RNA.
Embodiment 175. The composition of embodiment 174, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.
Embodiment 176 The composition of any one of embodiments 168-173, wherein the alteration comprises addition of a 5’ cap to the RNA.
Embodiment 177 The composition of embodiment 176, wherein the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment 178 The composition of any one of embodiments 168-177, wherein the second RNA comprises:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; or d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 179 A method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one of the compositions of embodiments 159-178.
Embodiment 180 The composition of any one of embodiments 159-178 for use in a method of treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis.
Embodiment 18 A composition comprising an RNA encoding an IL-12sc
composition comprising an RNA encoding a GM-CSF
composition comprising an RNA encoding an IFN ⁇ 2b
Embodiment 184 A composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24.
Embodiment 185 A composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9.
Embodiment 186 A composition comprising an RNA encoding an IL-12sc
RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18.
Embodiment 187 A composition comprising an RNA encoding a GM-CSF
RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29.
Embodiment 188 A composition comprising an RNA encoding an IFN ⁇ 2b
RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 189 A composition comprising an RNA encoding an IL-15 sushi protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.
Embodiment 190 A composition comprising an RNA encoding an IL-2 protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 191 A composition comprising any two of the following RNAs: a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 192 A composition comprising:
amino acid sequence of SEQ ID NO: 27 amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29.
Embodiment 193 A composition comprising: a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18; and b. an RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 194. A composition comprising:
Embodiment 195 A composition comprising:
Embodiment 196 A composition comprising:
nucleotides of SEQ ID NO: 27 amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and b. an RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.
Embodiment 197 A composition comprising: a. an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and b. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.
Embodiment 198 A composition comprising:
Embodiment 199 A composition comprising:
Embodiment 200 A composition comprising:
Embodiment 201 A composition comprising: a. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and b. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 202 A composition comprising any three of the following:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and a. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 203 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 204 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
nucleotides of SEQ ID NO: 27 amino acid sequence of SEQ ID NO: 27, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29; and c. an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.
Embodiment 205 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 206 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 207 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 208 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 209 A composition comprising:
Embodiment 210 A composition comprising:
Embodiment 211 A composition comprising:
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 212 A composition comprising:
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and c. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 213. A composition comprising any four of the following:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 214 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 215. A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
Embodiment 216 A composition comprising:
RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 217 A composition comprising: a. an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18;
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 218 A composition comprising:
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and d. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 219. A composition comprising:
RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23;
an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26; and e. an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9, and/or comprising nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
Embodiment 220 A pharmaceutical formulation comprising any one of the
compositions of embodiments 181-219 are compositions of embodiments 181-219.
Embodiment 22 A pharmaceutical formulation comprising any one of the
compositions of embodiments 181-219and a pharmaceutically acceptable excipient Embodiment 222.
Embodiment 223 The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of reducing the size of a tumor.
Embodiment 224 The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing the reoccurrence of cancer in remission.
Embodiment 225 The composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing cancer metastasis.
Embodiment 226 A method for treating or preventing cancer comprising
Embodiment 227 A method for reducing the size of a tumor comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
Embodiment 228 A method for preventing the reoccurrence of cancer in
remission comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
Embodiment 229. A method for preventing cancer metastasis comprising
Embodiment A A medical preparation comprising RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.
Embodiment A 2 The medical preparation of embodiment A 1, further comprising RNA encoding an IL-15 sushi protein.
Embodiment A 3 The medical preparation of embodiment A 1 or 2, further comprising RNA encoding an IL-2 protein.
Embodiment A 4 The medical preparation of any one of embodiments A 1 to 3, further comprising RNA encoding an IFN ⁇ protein.
Embodiment A 5 The medical preparation of embodiment A 4, wherein the IFN ⁇ protein is an IFN ⁇ 2b protein.
Embodiment A 6 The medical preparation of embodiment A 2, comprising RNA
RNA encoding an IL-12sc protein RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.
Embodiment A 7 The medical preparation of embodiment A 3, comprising RNA
RNA encoding an IL-12sc protein RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.
Embodiment A 8 The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN ⁇ protein.
Embodiment A 9 The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL- 15 sushi protein, and RNA encoding an IFN ⁇ protein.
Embodiment A 10. The medical preparation of embodiment A 4 or 5, comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL- 2 protein, and RNA encoding an IFN ⁇ protein.
Embodiment A 11 The medical preparation of any one of embodiments A 1-10, wherein (i) the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.
Embodiment A 12 The medical preparation of any one of embodiments A 1-11, wherein (i) the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM- CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
Embodiment A 13 The medical preparation of any one of embodiments A 2-12, wherein (i) the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL- 15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.
Embodiment A 14 The medical preparation of any one of embodiments A 3-13, wherein (i) the RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9.
Embodiment A 15 The medical preparation of any one of embodiments 4-14, wherein (i) the RNA encoding an IFN ⁇ protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFN ⁇ protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
Embodiment A 16 The medical preparation of any one of embodiments A 1-15, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment A 17 The medical preparation of any one of embodiments A 1-16, wherein each RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment A 18 The medical preparation of any one of embodiments A 1-17, wherein each RNA comprises a modified nucleobase in place of each uridine.
Embodiment A 19 The medical preparation of any one of embodiments A 16-18, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ) or 5- methyl-uridine (m5U).
the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ) or 5- methyl-uridine (m5U).
Embodiment A 20 The medical preparation of embodiment A 19, wherein the modified nucleobase is N1-methyl-pseudouridine (m1 ⁇ ).
Embodiment A 21 The medical preparation of any one of embodiments A 1-20, wherein at least one RNA comprises a 5’ cap.
Embodiment A 22 The medical preparation of any one of embodiments A 1-21, wherein each RNA comprises a 5’ cap.
Embodiment A 23 The medical preparation of embodiment A 21 or 22, wherein the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment A 24 The medical preparation of any one of embodiments A 1-23, wherein at least one RNA comprises a 5’ UTR.
Embodiment A 25 The medical preparation of any one of embodiments A 1-24, wherein each RNA comprises a 5’ UTR.
Embodiment A 26 The medical preparation of embodiment A 24 or 25, wherein the 5’ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.
Embodiment A 27 The medical preparation of any one of embodiments A 1-26, wherein at least one RNA comprises a 3’ UTR.
Embodiment A 28 The medical preparation of any one of embodiments A 1-27, wherein each RNA comprises a 3’ UTR.
Embodiment A 29 The medical preparation of embodiment A 27 or 28, wherein the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
Embodiment A 30 The medical preparation of any one of embodiments A 1-29, wherein at least one RNA comprises a poly-A tail.
Embodiment A 31 The medical preparation of any one of embodiments A 1-30, wherein each RNA comprises a poly-A tail.
Embodiment A 32 The medical preparation of embodiment A 30 or 31, wherein the poly- A tail comprises at least 100 nucleotides.
Embodiment A 33 The medical preparation of any one of embodiments A 1-32, wherein at least one RNA comprises a 5’ cap, a 5’ UTR, a 3’ UTR, and a poly-A tail.
Embodiment A 34 The medical preparation of any one of embodiments A 1-33, wherein each RNA comprises a 5’ cap, a 5’ UTR, a 3’ UTR, and a poly-A tail.
Embodiment A 35 The medical preparation of embodiment A 33 or 34, wherein a. the 5’ cap is m2 7,3’-O Gppp(m1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G;
the 5’ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6;
the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide
the poly-A tail comprises at least 100 nucleotides.
Embodiment A 36 The medical preparation of any one of embodiments A 1 to 35,
RNA is mRNA
Embodiment A 37 The medical preparation of any one of embodiments A 1 to 36, which comprises a further therapeutic agent.
Embodiment A 38 The medical preparation of embodiment A 37, wherein the further therapeutic agent is an anti-cancer therapeutic agent.
Embodiment A 39 The medical preparation of embodiment A 37 or 38, wherein the further therapeutic agent is a checkpoint modulator.
Embodiment A 40 The medical preparation of embodiment A 39, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.
Embodiment A 41 The medical preparation of any one of embodiments A 1 to 40, which is a kit comprising at least two containers, each container comprising at least one of said RNAs.
Embodiment A 42 The medical preparation of embodiment A 41, which comprises each RNA in a separate container.
Embodiment A 43 The medical preparation of embodiment A 41 or 42, wherein the further therapeutic agent is in a container not comprising the RNA.
Embodiment A 44 The medical preparation of any one of embodiments A 41-43, further comprising instructions for use of the medical preparation for treating or preventing cancer.
Embodiment A 45 The medical preparation of any one of embodiments A 1 to 40, which is a pharmaceutical composition comprising the RNAs.
Embodiment A 46 The medical preparation of embodiment A 45, wherein the
composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
Embodiment A 47 The medical preparation of any one of embodiments A 1 to 46,
RNA is present in a form selected from a liquid form, a solid form, or a combination thereof.
Embodiment A 48 The medical preparation of embodiment A 47, wherein the solid form is a frozen form or a dehydrated form.
Embodiment A 49 The medical preparation of embodiment A 48, wherein the dehydrated form is a freeze-dried or spray-dried form.
Embodiment A 50 The medical preparation of any one of embodiments A 1 to 49 for pharmaceutical use.
Embodiment A 51 The medical preparation of embodiment A 50, wherein the
pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.
Embodiment A 52 The medical preparation of any one of embodiments A 1 to 51 for use in a method for treating or preventing cancer.
Embodiment A 53 The medical preparation of any one of embodiments A 38-52, wherein the cancer is a sarcoma, carcinoma, or lymphoma.
Embodiment A 54 The medical preparation of any one of embodiments A 38-53, wherein the cancer is a solid tumor.
Embodiment A 55 The medical preparation of embodiment A 54, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
Embodiment A 56 The medical preparation of embodiment A 54 or 55, wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or
Embodiment A 57 The medical preparation of any one of embodiments A 1-56, wherein the RNA is for intra-tumoral or peri-tumoral administration.
Embodiment A 58 The medical preparation of any one of embodiments A 37-57, wherein the further therapeutic agent is for systemic administration.
Embodiment A 59 The medical preparation of any one of embodiments A 1-58, which is for administration to a human.
Embodiment A 60 The medical preparation of any one of embodiments A 44 and 47-59, wherein treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.
Embodiment B RNA for use in a method for treating or preventing cancer in a subject, wherein the method comprises administering RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.
Embodiment B 2 The RNA of Embodiment B 1, wherein the method further comprises administering RNA encoding an IL-15 sushi protein.
Embodiment B 3 The RNA of Embodiment B 1 or 2, wherein the method further
Embodiment B 4 The RNA of any one of embodiments B 1 to 3, wherein the method further comprises administering RNA encoding an IFN ⁇ protein.
Embodiment B 5 The RNA of Embodiment B 4, wherein the IFN ⁇ protein is an IFN ⁇ 2b protein.
Embodiment B 6 The RNA of Embodiment B 2, wherein the method comprises
RNA encoding an IL-12sc protein administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.
Embodiment B 7 The RNA of Embodiment B 3, wherein the method comprises
RNA encoding an IL-12sc protein administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.
Embodiment B 8 The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN ⁇ protein.
Embodiment B 9 The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFN ⁇ protein.
Embodiment B 10 The RNA of Embodiment B 4 or 5, wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-2 protein, and RNA encoding an IFN ⁇ protein.
Embodiment B 11 The RNA of any one of embodiments B 1-10, wherein (i) the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.
Embodiment B 12 The RNA of any one of embodiments B 1-11, wherein (i) the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
Embodiment B 13 the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of
RNA of any one of embodiments B 2-12 wherein (i) the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.
Embodiment B 14 The RNA of any one of embodiments B 3-13, wherein (i) the RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9.
Embodiment B 15 The RNA of any one of embodiments B 4-14, wherein (i) the RNA encoding an IFN ⁇ protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFN ⁇ protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
Embodiment B 16 The RNA of any one of embodiments B 1-15, wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment B 17 The RNA of any one of embodiments B 1-16, wherein each RNA comprises a modified nucleobase in place of at least one uridine.
Embodiment B 18 The RNA of any one of embodiments B 1-17, wherein each RNA comprises a modified nucleobase in place of each uridine.
Embodiment B 19 The RNA of any one of embodiments B 16-18, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ) or 5-methyl-uridine (m5U).
the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ) or 5-methyl-uridine (m5U).
Embodiment B 20 The RNA of Embodiment B 19, wherein the modified nucleobase is N1-methyl-pseudouridine (m1 ⁇ ).
Embodiment B 21 The RNA of any one of embodiments B 1-20, wherein at least one RNA comprises a 5’ cap.
Embodiment B 22 The RNA of any one of embodiments B 1-21, wherein each RNA comprises a 5’ cap.
Embodiment B 23 The RNA of Embodiment B 21 or 22, wherein the 5’ cap is m2 7,3’- O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G.
Embodiment B 24 The RNA of any one of embodiments B 1-23, wherein at least one RNA comprises a 5’ UTR.
Embodiment B 25 The RNA of any one of embodiments B 1-24, wherein each RNA
Embodiment B 26 The RNA of Embodiment B 24 or 25, wherein the 5’ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.
Embodiment B 27 The RNA of any one of embodiments B 1-26, wherein at least one RNA comprises a 3’ UTR.
Embodiment B 28 The RNA of any one of embodiments B 1-27, wherein each RNA
Embodiment B 29 The RNA of embodiment B 27 or 28, wherein the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
Embodiment B 30 The RNA of any one of embodiments B 1-29, wherein at least one RNA comprises a poly-A tail.
Embodiment B 31 The RNA of any one of embodiments B 1-30, wherein each RNA
Embodiment B 32 The RNA of embodiment B 30 or 31, wherein the poly-A tail
Embodiment B 33 The RNA of any one of embodiments B 1-32, wherein at least one RNA comprises a 5’ cap, a 5’ UTR, a 3’ UTR, and a poly-A tail.
Embodiment B 34 The RNA of any one of embodiments B 1-33, wherein each RNA
Embodiment B 35 The RNA of embodiment B 33 or 34, wherein
the 5’ cap is m 2 7,3’-O Gppp(m 1 2’-O )ApG or 3 ⁇ -O-Me-m 7 G(5')ppp(5')G;
the 5’ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6;
the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide
the poly-A tail comprises at least 100 nucleotides.
Embodiment B 36 The RNA of any one of embodiments B 1 to 35, wherein the RNA is mRNA.
Embodiment B 37 The RNA of any one of embodiments B 1 to 36, wherein the method further comprises administering a further therapy.
Embodiment B 38 The RNA of embodiment B 37, wherein the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) immunotherapy, (iii) radiotherapy, and (iv) chemotherapy.
Embodiment B 39 The RNA of embodiment B 37 or 38, wherein the further therapy comprises administering a further therapeutic agent.
Embodiment B 40 The RNA of embodiment B 39, wherein the further therapeutic agent is an anti-cancer therapeutic agent.
Embodiment B 41 The RNA of embodiment B 39 or 40, wherein the further therapeutic agent is a checkpoint modulator.
Embodiment B 42 The RNA of embodiment B 41, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.
Embodiment B 43 The RNA of any one of embodiments B 1-42, wherein the cancer is a sarcoma, carcinoma, or lymphoma.
Embodiment B 44 The RNA of any one of embodiments B 1-43, wherein the cancer is a solid tumor.
Embodiment B 45 The RNA of embodiment B 44, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
Embodiment B 46 The RNA of embodiment B 44 or 45, wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
HL Hodgkin lymphoma
non-Hodgkin lymphoma prostate tumor
ovarian tumor renal cell tumor
renal cell tumor gastrointestinal tract tumor
hepatic tumor colorectal tumor
tumor with vasculature me
Embodiment B 47 The RNA of any one of embodiments B 1-46, wherein the RNA is administered intra-tumorally or peri-tumorally.
Embodiment B 48 The RNA of any one of embodiments B 39 to 47, wherein the further therapeutic agent is administered systemically.
Embodiment B 49 The RNA of any one of embodiments B 1-48, wherein the subject is a human.
Embodiment B 50 The RNA of any one of embodiments B 1-49, wherein the RNAs are administered at the same time.
Embodiment B 51 The RNA of any one of embodiments B 1-50, wherein the RNAs are administered by administering a composition comprising a combination of the RNAs.
Embodiment B 52 The RNA of any one of embodiments B 1-49, wherein at least two of the RNAs are administered at different times.
Embodiment B 53 The RNA of any one of embodiments B 1-49 and 52, wherein the RNAs are administered by administering at least two compositions, each composition comprising at least one of said RNAs.
Embodiment B 54 The RNA of any one of embodiments B 1 to 53, wherein treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.
Embodiment B 55 The RNA of any one of embodiments B 1 to 54, which is or comprises one or more of the RNAs administered in said method.
Embodiment B 56 The RNA of embodiment B 55, which is or comprises one or more selected from the group consisting of the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN ⁇ protein.
Embodiment B 57 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein.
Embodiment B 58 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding a GM-CSF protein.
Embodiment B 59 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-15 sushi protein.
Embodiment B 60 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-2 protein.
Embodiment B 61 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IFN ⁇ protein.
Embodiment B 62 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein and the RNA encoding a GM-CSF protein.
Embodiment B 63 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-15 sushi protein.
Embodiment B 64 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-2 protein.
Embodiment B 65 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IFN ⁇ protein.
Embodiment B 66 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, and the RNA encoding an IFN ⁇ protein.
Embodiment B 67 The RNA of embodiment B 55 or 56, which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN ⁇ protein.
FIGS 1A– 1G shows results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 8, 10, 12, 14 and individual tumor growth was monitored to day 41.
FIG 1A and FIG 1D show results when using IL-2, IL- 12sc, and GM-CSF (ModA) mRNA.
FIG 1B and FIG 1E show IL-2, IL-12sc, and GM-CSF (ModB) mRNA.
FIG 1C and FIG 1F show results when using luciferase mRNA (ModA).
FIGS 2A– 2D show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 19, 21, 24, 26, 28 and 31 and individual tumor growth was monitored to day 48.
FIG 2A shows GM-CSF, IL-2, IL-12sc (ModA).
FIG 2B shows GM-CSF, IL-2, IL-12sc (ModB).
FIG 2C shows luciferase mRNA (ModA) as a control.
FIG 2D shows Ringer’s solution as a control.
FIGS 3A– 3C show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 13, 15, 18, 20 and 22 and tumor growth was monitored to day 42.
FIG 3A shows IL-2, GM-CSF, IL-12sc (ModB).
FIG 3B shows IL-15 sushi, GM-CSF, IL-12sc (ModB).
FIG 3C shows luciferase mRNA (ModB) as a control.
FIGS 4A– 4F show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45.
FIG 4A shows IL-2, IL-12sc, and GM- CSF (ModA).
FIG 4B is a duplicate in the same experiment as described in FIG 5A, showing IL-2, IL-12sc, and GM-CSF (ModB).
FIG 4C shows IL-15 sushi, IL-12sc, and GM-CSF (ModA).
FIG 4D is a duplicate in the same experiment as described in FIG 5B, showing IL- 15 sushi, IL-12sc, and GM-CSF (ModB).
FIG 4E shows control luciferase mRNA (ModA).
FIGS 5A– 5D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45.
FIG 5A is a duplicate in the same experiment as described in FIG 4B, showing IL-2, IL-12sc, and GM-CSF (ModB).
FIG 5B is a duplicate in the same experiment as described in FIG 4D, showing IL-15 sushi, IL-12sc, and GM-CSF (ModB).
FIG 5C is a duplicate in the same experiment as described in FIG 6C, showing IL-2, IL-12sc, GM-CSF, and IFN ⁇ (ModB).
FIGS 6A and 6B show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted.
FIG 6A is a duplicate in the same experiment as described in FIG 7A, showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
FIG 6B is a duplicate in the same experiment as described in FIG 7C, showing luciferase mRNA (ModB).
N 8 mice/group.
FIGS 6C and 6D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 11, 13, 15, 17 and individual tumor growth was plotted.
FIG 6C is a duplicate in the same experiment as described in FIG 5C, showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
FIG 6D is a duplicate in the same experiment as described in FIG 4F and 5D, showing luciferase mRNA (ModB).
N 8 mice/group.
FIG 6E and 6F show results of experiments where MC38 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 15, 19, 23 and individual tumor growth was plotted.
FIG 6E shows GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
FIGS 7A– 7F show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted.
FIG 7A is a duplicate in the same experiment as described in FIG 6A, showing IL-2, IL-12sc, GM-CSF, IFN ⁇ (ModB).
FIG 7B shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
FIG 7C is a duplicate in the same experiment as described in FIG 6B, showing a luciferase mRNA (ModB) control.
FIG 7D is a duplicate of the same experiment as described in FIG 9A, showing IL-2, IL-12sc, GM- CSF, IFN ⁇ (ModB).
FIG 7E shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
FIG 7F is a duplicate of the same experiment as described in FIG 9F, showing a luciferase mRNA (ModB) control.
FIGS 8A– 8H show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 12, 15, 19 and 22 and individual tumor growth was monitored and plotted to day 35.
FIG 8A shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
FIG 8B shows IL-15 sushi, IL-12sc, IFN ⁇ (ModB).
FIG 8C shows IL-15 sushi, GM-CSF, IFN ⁇ (ModB).
FIG 8D shows GM-CSF, IL-12sc, IFN ⁇ (ModB).
FIG 8E shows IL-15 sushi, GM-CSF, IL-12sc (ModB).
FIG 8F shows a luciferase mRNA (ModB) control.
FIGS 8G and 8H show tumor growth kinetics of the study shown in FIGS 8A-8F.
FIG 8G shows mean tumor volumes up to day 33 for all treatment groups.
FIG 8H shows tumor growth repression.
T/C Tumor/Control based on mean tumor volume
FIGS 9A– 9F show experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 19, 21, 23, 26, 28 and 30 and tumor growth was monitored to day 50.
FIG 9A is a duplicate in the same experiment as described in FIG 7D, showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
FIG 9B shows IL-2, IL-12sc, IFN ⁇ (ModB).
FIG 9C shows GM-CSF, IL-2, IFN ⁇ (ModB).
FIG 9D shows GM-CSF, IL-12sc, IFN ⁇ (ModB).
FIG 9E shows GM-CSF, IL-2, IL-12sc (ModB).
FIGS 10A– 10B shows tumor growth kinetics of the study shown in Figure 9.
FIG 10A shows mean tumor volumes up to day 36 for all treatment groups.
FIG 10B shows tumor growth repression.
T/C Tumor/Control based on mean tumor volume
FIG 11 shows a bar graph of data from the experiments shown in FIG 9 showing mRNA mixtures with significant reduction in tumor volume, where the number of mice in each of the treatment groups with significant tumor reduction was compared to the luciferase control group based on Z score of tumor volume and the ratio between tumor volume change and the mean of the control group.
FIGS 12A– 12D show the results of experiments where mice that were 1) tumor na ⁇ ve, or 2) had been previously injected subcutaneously with 5x10 5 B16F10 cells and rejected the original tumor following intratumoral cytokine mRNA treatment. Both groups were re-challenged with B16F10 tumors.
FIG 12A shows tumor na ⁇ ve host mice.
FIG 12B shows mice that had previously rejected B16F10 tumors following intratumoral cytokine mRNA treatment with GM-CSF, IL-15sushi, IL-12sc, IFN ⁇ (ModB). Mice were monitored for 55 days following B16F10 injection and tumor growth for each mouse was plotted.
FIG 12A shows an example of localized vitiligo at the tumor site.
FIG 12D shows the results of experiments where mice that were tumor na ⁇ ve (triangle symbol), or had been previously injected subcutaneously with CT26 tumor cells and rejected the original tumor following intratumoral cytokine mRNA treatment (circle symbol).
CT26-WT CT26 tumor cells
CT26- ⁇ gp70 tumor cells CT26- ⁇ gp70 tumor cells
Mice were monitored for 21 days following tumor cell injection. All nine but one na ⁇ ve mice engrafted with CT26-WT cells and all na ⁇ ve mice engrafted with CT26- ⁇ gp70 cells developed tumors, whereas all three tumor-free mice rejected the CT26 tumor cells and did not exhibit growth of CT26 and CT26- ⁇ gp70 tumors, respectively.
ModB cytokine mRNA IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇
ModB control mRNA luciferase
FIGS 14A– 14F show results of experiments where human HEK293 (FIG 14B) and melanoma cell lines (A101D (FIG 14C), A2058 (FIG 14D), A375 (FIG 14E), and Hs294T (FIG 14F)) were transfected with human cytokine mRNA mixture (IL-12sc, GM- CSF, IL-15 sushi and IFN ⁇ 2b) in a range of mRNA doses. Supernatants were collected 24 hrs after transfection and protein concentrations were determined with cytokine specific ELISAs.
FIG 14A shows a schematic of the experiment.
FIGS 16A– 16E show the results of experiments where immune compromised mice bearing human A375 tumor xenografts received a single injection with the ModB mRNA mixture encoding the human cytokines (IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b; “the IL15 sushi mixture”) or (IL-2, IL-12sc, GM-CSF and IFN ⁇ 2b;“the IL2 mixture”).
FIG 16A shows IFN ⁇ 2b
FIG 16B shows IL-2
FIG 16C shows IL-12sc
FIG 16D shows IL-15 sushi
FIG 16E shows GM-CSF.
FIGS 17A– 17C show the results of experiments where mRNA was isolated from A375 tumors at 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, and 72 hrs after injection of ModB cytokine mRNA mixture (IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ 2b) or (IL-2, IL-12sc, GM- CSF, IFN ⁇ 2b). Expression of interferon alpha response genes were monitored by qPCR.
FIG 17A shows human ISG15
FIG 17B shows human ISG54
FIG 17C shows human MX1.
FIGS 18A– 18E show the results of experiments where mice were implanted with B16F10 tumor cells and treated with mRNA mixtures (FLT3L, IL-2, 41BBL, and CD27L-CD40L) with or without IFN ⁇ .
mRNA mixtures without IFN ⁇ in standard (ModA, FIG 18B) and modified forms (ModB, FIG 18C) were compared to those including IFN ⁇ in standard (ModA, FIG 18D) and modified forms (ModB, FIG 18E).
FIG 18A is a negative control where Ringer’s media without mRNA was provided.
FIGS 19A– 19E show the results of experiments where mice were implanted with tumors on one flank and received an IV injection of luciferase-expressing tumor cells that homed to the lung (FIG 19A). Mice in the treatment group received intratumoral injections of mRNA mixtures IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ into the flank tumor only while tumors in the lung were untreated.
FIG 19B shows exemplarily bioluminescence measurements in lungs and pictures of the according lungs taken out on the same day (day 20); tumor nodes are visual as black marks;
FIG 19C shows mean tumor volume of flank tumors as determined by caliper measurements;
FIG 19D shows total flux analysis of bioluminescence measurements on day 20;
FIG 19E shows lung weights.
FIGS 20A– 20G show the results of experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic administration of antibodies in dual flank tumor models.
Mice implanted with either the B16F10 tumor on the left and right flank or MC38 tumors on the left and right flank received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (Mod B) into only one flank tumor, while the other flank tumor was untreated. Mice also received intraperitoneal (systemic) injection of an anti-PD1 antibody.
FIG 20 shows overall survival in the B16F10 (FIG 20A) and MC38 (FIG 20B) tumor models.
FIGS 20C-G show the results of an experiment evaluating the anti-PD-1 antibody where mice were implanted with B16F10 tumors on one flank and received an IV injection of luciferase-expressing B16F10 tumor cells that homed to the lung. Mice received three intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ and also received three intraperitoneal (systemic) injection of an anti-PD-1 antibody. Tumor growth of the SC tumors is depicted in FIG 20C-F.
FIG 20C shows control mRNA and control antibody
FIG 20D shows control mRNA plus anti-PD1 antibody
FIG 20E shows cytokine mRNA mixture plus isotype control antibody.
FIG 20F shows cytokine mRNA plus anti-PD-1 antibody.
FIG 20G shows percent survival of all four treatment groups until day 70 after IV tumor inoculation; the treatment group that received mRNA plus anti-PD-1 antibody showed strongest anti-tumoral activity with 6 out of 15 mice being tumor-free on day 40 after tumor inoculation.
FIGS 21A– 21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic
FIG 21A shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 12 out of 16 mice being tumor-free on day 55 after tumor inoculation.
FIG 21B shows cytokine mRNA mixture plus isotype control antibody;
FIG 21C shows control mRNA plus anti-CTLA-4 antibody;
FIG 21D shows control mRNA and control antibody.
FIGS 21E-21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with anti- CTLA-4 antibody in B16F10 tumor model.
Mice bearing B16F10 tumors received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ . Mice also received intraperitoneal (systemic) injection of an anti-CTLA-4 antibody.
FIG 21E shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti- CTLA-4 resulted in strongest anti-tumoral activity with 6 out of 9 mice being tumor-free on day 70 after tumor inoculation.
FIG 21F shows cytokine mRNA mixture plus isotype control antibody
FIG 21G shows control mRNA plus anti-CTLA-4 antibody
FIG 21H shows control mRNA and control antibody.
FIG 21I shows percent survival of all four treatment groups until day 70 after tumor inoculation.
FIGS 22A– 22D shows the results of experiments designed to evaluate the effect of intratumoral injection of cytokine mRNA (Mod B) in human tumor xenografts of different human cancers. Intratumoral expression of each of the 4 mRNA encoded cytokines is shown: IL-12sc (FIG 22A), IFN ⁇ 2b (FIG 22B), GM-CSF (FIG 22C), and IL-15 sushi (FIG 22D).
FIGS 23A– 23D show the results of experiments designed to evaluate the effect of different intratumoral mRNA doses on the expression of the encoded cytokines: IL-15 sushi (FIG 23A), IL-12sc (FIG 23B), GM-CSF (FIG 23C) and IFN ⁇ 2b (FIG 23D).
FIGS 24A– 24G show the results of experiments where mice were implanted with B16F10 tumor, and treated with four intratumoral injections of cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB) or mRNA encoding a single cytokine. Tumor volume out to approximately day 70 was measured.
FIG 24A shows luciferase control;
FIG 24B shows the four-cytokine mixture;
FIG 24C shows IL-12sc mRNA only;
FIG 24D shows GM-CSF mRNA only;
FIG 24E shows IFN ⁇ mRNA only; and
FIG 24F shows IL-15 sushi only.
FIG 24G shows overall survival of B16F10 tumors treated with cytokine mRNA mixture or individual mRNA encoded cytokines. Survival data is from experiment presented in Figure 24A-F.
FIG 25 shows CD8+ immune cell infiltrate in subcutaneous tumors after control mRNA (“placebo”) and cytokine mRNA treatment.
FIGS 26A– 26C show results of measurements of CD8+ T cells specific for the gp70 tumor antigen of gp70 in blood of CT26 tumor bearing mice that had received intratumoral administration of cytokine mRNA treatment and control mRNA, respectively.
FIG 26C shows exemplarily a FACS histogram of CD8+ T cells stained with anti-mouse CD8 antibody and with the gp70-specific tetramer derived from an animal that had received control mRNA and
FIG 26B shows the example from one animal treated with cytokine mRNA.
FIG26C shows the analysis of percentage of gp70-specific CD8+ T-cells in blood 13 days after treatment start from 9 mice that had received four injections of control mRNA and 10 mice that had received 4 injections of cytokine mRNA.
FIGS 27A-27C show experiments where mice bearing B16F10 tumors on the left and right flanks received a single intratumoral mRNA injection with cytokine mRNA or control mRNA in only one tumor. On day 7 following the mRNA injection the left and right tumors were collected and subjected to RNA sequencing.
FIG 27B shows the results of ingenuity pathway analysis comparing the gene expression changes between the cytokine mRNA treatment vs control mRNA treated tumors.
Causal network analysis for treated tumor side (Column 1) and untreated tumor side (Column 2) was performed and Activation Z score (Top half) and Inhibition Z score (Lower half) was analyzed to define pathways up and down regulated, respectively.
FIG 27 shows cluster analysis of injected and non-injected tumors was performed based on 327 interferon gamma regulated genes. Both the injected and non- injected tumors of mice treated with cytokine mRNA showed upregulation of multiple IFN gamma genes in comparison to mice treated with control mRNA.
FIGS 28A– 28D show fluorescence micrographs of cells from a B16F10 dualtumor model.
Panel A shows the injected tumor treated with cytokine mRNA and panel B shows the corresponding uninjected tumor.
Panel C shows the injected tumor treated with control mRNA and panel D shows the corresponding uninjected tumor.
the slides were stained for CD4+, CD8+, and FoxP3+ cells.
FIGS 28E-G show frequency of CD4+, CD8+ and FOXP3+ cells quantified in the immunofluorescent images.
the frequency of CD4+ and CD8+ cells/mm 2 is presented in FIG 28E and 28F.
the ratio of the CD8+ frequency divided by FOXP3+ frequency is presented in FIG 28G.
FIGS 29A– 29G show mice with a single B16F10 tumor received a single injection with either mRNA encoding the Thy1.1 cell surface protein or vehicle alone (Ringer’s solution). At approximately 16-18 hours following intratumoral injection the tumor was excised, digested, stained with a panel of antibodies and analyzed by flow cytometry. The cell type and frequency of cells expressing Thy1.1 were characterized.
FIGS 30A– 30F show expression of the indicated proteins following various doses of cytokine mRNA or luciferase control mRNA detected in tumor lysates as described in Example 15.“IFNy” in FIG 30E indicates IFN ⁇ .
FIGS 31A– 31B show flow cytometry results for CD8+ and FOXP3+ (Treg) cells following control or cytokine mRNA treatments as described in Example 15. The observed ratio of CD8+ to Treg cells is shown in each panel.
FIGS 31C– 31D show flow cytometry results for polyfunctional CD8+ T cells following control or cytokine mRNA treatments as described in Example 15. The proportion of polyfunctional CD8+ T cells is shown in each panel.
FIG 31E shows the level of PD-L1 on infiltrating myeloid cells following control or cytokine mRNA treatments as described in Example 15.
FIG 31F shows the level of PD-1 on infiltrating CD8+ cells following control or cytokine mRNA treatments as described in Example 15.
FIGS 31G-H show the frequency of intratumoral Granzyme B CD8+ T cells following control or cytokine mRNA treatments as described in Example 15.
FIGS 32A– 32B show luciferase expression in various tissues following intratumoral injection of 50 ⁇ g mRNA encoding firefly luciferase as described in Example 16.
FIGS 33 shows data relating to an experiment essentially as shown in Figure 12.
FIG 34 shows the effect of depleting CD8+ T cells, CD4+ T cells or NK cells before treatment with cytokine mRNAs on survival in mice bearing B16F10 tumors as described in Example 17.
FIG 35 shows survival of WT and IFN ⁇ KO mice implanted with B16F10 tumor cells as described in Example 1 and treated with control or cytokine mRNAs as described in Example 18. DESCRIPTION OF THE SEQUENCES
Tables 1 and 2 provide a listing of certain sequences referenced herein.

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PCT/US2018/019878 2017-02-28 2018-02-27 Therapeutic rna WO2018160540A1 (en)

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BR112019017743A BR112019017743A2 (pt)	2017-02-28	2018-02-27	rna terapêutico
PE2019001764A PE20200735A1 (es)	2017-02-28	2018-02-27	Arn terapeutico
AU2018229278A AU2018229278A1 (en)	2017-02-28	2018-02-27	Therapeutic RNA
JP2019546924A JP6949131B2 (ja)	2017-02-28	2018-02-27	治療用ｒｎａ
MX2019010269A MX2019010269A (es)	2017-02-28	2018-02-27	Arn terapeutico.
SG11201907846VA SG11201907846VA (en)	2017-02-28	2018-02-27	Therapeutic rna
EP18709243.2A EP3589308A1 (en)	2017-02-28	2018-02-27	Therapeutic rna
CA3054733A CA3054733A1 (en)	2017-02-28	2018-02-27	Therapeutic rna
CN201880013857.3A CN110337305A (zh)	2017-02-28	2018-02-27	治疗性rna
KR1020197028213A KR102700956B1 (ko)	2017-02-28	2018-02-27	치료적 rna
EA201992022A EA201992022A1 (ru)	2017-12-12	2018-02-27	Терапевтическая рнк
CR20190444A CR20190444A (es)	2017-02-28	2018-02-27	Arn terapéutico
KR1020247028726A KR20240144261A (ko)	2017-02-28	2018-02-27	치료적 rna
IL26889419A IL268894A (he)	2017-02-28	2019-08-25	Rna רפואי
US16/552,248 US20200147176A1 (en)	2017-02-28	2019-08-27	Therapeutic RNA
PH12019501959A PH12019501959A1 (en)	2017-02-28	2019-08-27	Therapeutic rna
CONC2019/0010355A CO2019010355A2 (es)	2017-02-28	2019-09-25	Arn terapéutico
US17/245,605 US11865159B2 (en)	2017-02-28	2021-04-30	Therapeutic RNA
US18/516,006 US20240173382A1 (en)	2017-02-28	2023-11-21	Therapeutic RNA

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US62/464,981		2017-02-28
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BR112019017743A2 (pt)	2020-04-07
US20210290730A1 (en)	2021-09-23
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US20200147176A1 (en)	2020-05-14
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PE20200735A1 (es)	2020-07-23
US20240173382A1 (en)	2024-05-30
AU2018229278A1 (en)	2019-10-17
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